In the human body two hormones, parathormone and calcitonin, along with vitamin D have major roles in controlling the metabolism of calcium (Ca.sup.++) and phosphate (Pi). Binding of parathormone to kidney cells yields among other things increased renal tubular reabsorption of Ca.sup.++ and Mg.sup.++, and enhanced excretion of Pi. The rates of renal gluconeogenesis and renal respiration are increased and hydroxylation of vitamin D to its active form (1,25-dihydroxy-vitamin D) is increased. In bones, parathormone inhibits collagen synthesis in active osteoblasts, increases osteocytic and osteoclastic osteolysis and increases the rate of maturation of osteoblasts and osteoclasts. There is increased mobilization of Ca.sup.++ from bone and increased transport of Ca.sup.++ across the intestine, accompanied by elevation of plasma Ca.sup.++. Calcitonin reduces bone resorption, perhaps by inhibiting osteocytes and osteoclasts. This is accompanied by hypocalcemia and hypophosphatemia. Enhanced excretion of Pi may be a secondary response to alterations in plasma Ca.sup.++. Secretion of parathormone is reduced and of calcitonin is increased in response to elevated Ca.sup.++ in the circulation.
In the presence of calcitonin, Ca.sup.++ and Pi are deposited in bone. The exact identities of the intermediates involved in the early steps of mineral deposition are still somewhat unsettled. It appears that brushite, CaHPO.sub.4.2H.sub.2 O, is deposited first but then redissolves and is converted to amorphous calcium phosphate, a noncrystalline association of ions. This subsequently is believed to yield octacalcium phosphate, Ca.sub.8 (HPO.sub.4).sub.2 (PO.sub.4).sub.4.5H.sub.2 O, which in turn converts to hydroxyapatite, Ca.sub.10 (OH).sub.2 (PO.sub.4).sub.6, the least soluble mineral of the group.
Several pathological conditions in which bone mineralization is deficient have been observed. These can arise from a number of conditions including, for example, abnormalities in hormonal regulation, renal cortical damage and dietary Ca.sup.++ and phosphate deprivation. Two specific examples are given for illustration.
a. Osteoporosis
Osteoporosis is characterized by reduced mineral content of bone, especially trabecular bone, and is particularly prevalent in postmenopausal women. The underlying causes of osteoporosis in the female appear first to arise at about the age of 30-40 years with reduced secretion of calcitonin. This is accompanied by hypocalcemia and subsequent elevation of parathormone. The 1 alpha-hydroxylase activity of the kidney becomes elevated with a probable resulting increase in "remodeling" of mineral deposits, especially in trabecular bone. The skeletal complications of osteoporosis become apparent after the secretion of estrogen begins to decrease (i.e. 40-50 years of age). The metabolism of vitamin D appears to be reduced when the estrogen levels are lower, and the intestinal absorption of calcium is affected. It would appear that the reduced intestinal absorption of calcium places an increased demand on the skeletal system, which in time yields reduced mineral deposits in trabecular bone. This results, for example, in an increased incidence of fractures of the hip and vertebrae. Similar demineralization also can arise from hyperparathyroidism and chronic renal insufficiency.
b. X-linked hypophosphatemia
X-linked hypophosphatemia is the most common form of vitamin D-resistant rickets. Human patients with this disease have reduced renal tubular reabsorption of phosphate, low plasma phosphate and resistance to 1,25-dihydroxy-vitamin D administration. These patients also exhibit osteomalacic bone disease (i.e. softening of the bone due to impaired mineralization with excess accumulation of osteoid).
There is at the present time a clinical need for a rapid assay of mineral content of bone samples. Procedures such as scanning electron microscopy and X-ray crystallography have been used experimentally, but these techniques are cumbersome and time-consuming to perform. Furthermore, the results from these two techniques have not been in full agreement. Wet chemical techniques of bone analysis also have serious limitations. Ashing and extraction normally are prerequisites to elemental analysis, and these can result in incomplete recovery of individual components. Furthermore, total phosphate analyses are seriously affected by procedures used routinely in the clinical setting to treat patients. Thus the results of phosphate analyses do not necessarily yield a reliable indication of variations in bone mineralization. A more reliable approach would be to identify and quantify mineral forms in unfractionated bone biopsy samples.